The CB1 cannabinoid receptor is a target for pharmaceutical drug design for neuroprotection in stroke, muscle relaxation in spastic disorders, analgesia, metabolic energy balance, and substance abuse disorders. However, unwanted side effects, such as cognitive and memory dysfunction, emotional disorders, and sedation, have curtailed development of CB1 ligands for therapeutic purposes. CB1 receptors regulate signal transduction pathways that modulate neuronal functions intrinsic to neuroprotection, synaptic plasticity, and neurotransmitter release. The working hypothesis is that CB1 receptor agonist and antagonist ligands exert their influence on the juxtamembrane C-terminal domain to initiate key signal transduction pathways in neuronal cells; and that this signaling can be modulated by palmitoylation, phosphorylation, and protein association at the juxtamembrane C-terminal domain. The proposed research will elucidate mechanisms by which CB1 ligands trigger receptor-Gi/o protein activation of signal transduction via the effectors adenylyl cyclase and mitogen activated protein kinase (MAPK), leading to phosphorylation pathways regulated by protein kinase A (PKA) and ERK1/2. The aims are to: 1. Characterize the signal transduction dependence on the juxtamembrane C-terminal domain. The cAMP/PKA and ERK responses to CB1 agonists having diverse structure and efficacy will be investigated in CB1 (L7.60(404)I,F) mutants stably expressed in HEK293 fibroblast and SH-SY5Y neuronal cells. 2. Characterize the effects of palmitoylation at the juxtamembrane C-terminal on CB1 receptor function by determining agonist-evoked responses in a C7.71(415)S CB1 mutant stably expressed in HEK293 and SH- SY5Y cells. 3. Characterize the effects of phosphorylation at the juxtamembrane C-terminal Ser/Thr on CB1 receptor function by determining agonist-evoked responses in phosphorylation-deficient (S401A, S410A, S414A) and phosphorylation-mimics (S401D, S410D, S414D) CB1 mutants stably expressed in SH-SY5Y cells. 4. Characterize the regulation of signal transduction via GASP1 interaction with the juxtamembrane C-terminal H8 domain of the CB1 receptor by examining the influence of juxtamembrane C-terminal disruption (L7.60(404)I,F), palmitoylation, and phosphorylation on CB1-GASP1a protein associations will be examined. The results of the proposed research will advance our understanding of biochemical mechanisms by which CB1 orthosteric ligands activate neuronal signaling responses. This understanding is critical to the design of drugs targeting the beneficial clinical responses with minimal undesirable effects.